Brain (1973) 96, 235-246
AXOPLASMIC FLOW IN AXONAL NEUROPATHIES
I. AXOPLASMIC FLOW IN CATS WITH TOXIC NEUROPATHIES
W. G. BRADLEY AND M. H. WILLIAMS
(From the Muscular Dystrophy Group Research Laboratories, Newcastle General Hospital, and
Department of Neurology, the University of Newcastle upon Tyne)
INTRODUCTION
THE passage of large amounts of protein and other compounds from the nerve
cell body down the axon is a widely accepted phenomenon to which the term
axoplasmic flow has been applied (see Barondes, 1967). Substances may also travel
in a retrograde fashion from the periphery of the axon to the neuron (Lubinska,
Niemierko and Zelena, 1963; Kerkut, Shapira and Walker, 1967; Watson, 1968;
Kristensson, 1970; Kristensson, Olsson and Sjostrand, 1971; Kristensson and
Olsson, 1971), though the amount is probably less than the orthograde flow
(Lubinska et al, 1963; Lubinska, 1971; Edstrom and Mattsson, 1972). Many
reports have described material flowing in an orthograde fashion from the neuron
to the periphery of the axon at two main rates, the slow at about 1 to 2 mm/day,
and the fast at several hundred mm/day. There are, however, reports of many
intermediate velocities and this matter has been recently reviewed (Bradley,
Murchison and Day, 1971).
The function of the axoplasmic flow material is not known, though at least five possibilities
might be mentioned:
(1) The provision of enzymes and precursors for the distal synthesis of transmitter substances.
Catecholamine granules (Dahlstrom and Haggendal, 1966) and choline acetyl-transferase (Frizell,
Hasselgren and Sjostrand, 1970) are both synthesized in the neuron and pass down the axon.
(2) The provision of "trophic factors" which have been supposed to be responsible for the many
effects of the presence of an intact nerve supply upon an innervated structure. Proteins released
from the neurohypophysis with the octopeptide hormones (neurophysins: Hope and Uttenthal,
1968; Dean and Hope, 1968; Geffen, Livett and Rush, 1969) and from the sympathetic nerves and
adrenal medulla with catecholamines (chromogranins and dopamine ^3-oxidase: Viveros, Arqueros
and Kirshner, 1968; De Potter, de Shaepdryver, Moerman and Smith, 1969; Kirshner, 1970),
and the trans-synaptic passage of material (Grafstein, 1971) may be examples of this phenomenon.
(3) The transmission of information within the extremely elongate cell which is the neuron
(Grafstein, 1969). For instance, a message is required to trigger the degeneration of the distal
parts of the axon and the axonal reaction of the nerve cell body following axonal section, and this
message may be conveyed by axoplasmic flow.
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BY
236
W. G. BRADLEY AND M. H. WILLIAMS
(4) The transfer of information or trophic factors to Schwann cells. Singer and Salpeter (1966a
and b) have suggested that material is transported from the Schwann cells to the axons. Similarly
a reversed passage of trophic influence must be considered to exist if the theory of secondary
segmental demyelination advanced by Dyck (1973) were correct.
(5) The provision of nutrients for the maintenance of the distal terminals of nerves.
MATERIAL AND METHODS
Young cats (1-5-4-25 kg body weight) of mixed breed were studied. Some animals were treated
with vincristine, acrylamide or TOCP to induce a toxic neuropathy of mild to moderate severity.
Animals receiving neurotoxins were regularly examined for general well-being, gait, ability to
stand from lying, and reaction to pin-prick on the feet. The dosage of drugs was adjusted to induce
mild to moderate ataxia of gait and weakness of the hind-limbs in two to six weeks. The dosages
used were: vincristine 0-04 mg/kg body weight/twice weekly intramuscularly; acrylamide 20
mg/kg body weight/day five days/week orally; TOCP (Coalite and Chemical Products Ltd.,
Bolsover, Derbyshire), 0-25 mg/kg body weight every two weeks subcutaneousry.
Dorsal root ganglion injection.—Control animals, and those with mild to moderate signs of a
neuropathy received an injection of L-leucine-4,5-3H into the seventh lumbar dorsal root ganglion
on one side. The method was similar to that of Ochs and Ranish (1969). Cats were anesthetized
with pentobarbitone (30 mg/kg body weight) and halothane in oxygen by face mask. Under sterile
conditions, the sixth and seventh lumbar and first sacral vertebrae were exposed, the lamkue on one
side removed, revealing the seventh lumbar dorsal root ganglion. Under a dissecting microscope,
a glass micropipette of tip external diameter 50-70 |x was introduced through the capsule after
puncture with afinesteel needle. The micropipette was lowered to a depth of 0-5 mm (Lasek, 1968)
with a micromanipulator. The injection volume was measured with a Hamilton 100 [il syringe
(A. V. Howe & Co. Ltd., No. 701) and a volume of 15 [^'(equivalent to 15 nCi of L-leucine-4,5-aH;
specific activity 17 Ci/mmol, 1 mCi/ml; Radiochemical Centre, Amersham, Bucks) was delivered
slowly over three minutes. Any leaking radioactive solution was absorbed by lint. The laminectomy
Downloaded from http://brain.oxfordjournals.org/ by John Lyftogt on September 11, 2013
It has been suggested that axonal neuropathies of the "dying back" type (Cavanagh,
1964) might result from impairment of this fifth possible function of axoplasmic
fiow,'with a decrease of the transport of materials, and in particular of protein from
the nerve cell body to the peripheral branches of the axon (see inter alii Bradley,
Lassman, Pearce and Walton, 1970). Pleasure, Mishler and Engel (1969) reported an
experiment to test this hypothesis, studying the movement of radioactive-labelled
protein in the dorsal and ventral roots of cats with acrylamide and triorthocresyl
phosphate (TOCP) neuropathy. The slower rates of axoplasmic flow only were
studied by virtue of the time points chosen for investigation. They found evidence of
axoplasmic flow in all situations, except in dorsal roots in acrylamide intoxication,
where flow appeared absent. The interpretation was that acrylamide caused axonal
degeneration in sensory fibres by damaging the slow rates of axoplasmicflow,and
that the mechanism of axoplasmic flow in TOCP neuropathy must be different.
Bradley et al. (1970) later suggested that the slower components of axoplasmic flow
might be impaired in "dying back" neuropathies, and that faster rates offlowmight
be impaired in neuropathies such as that of vincristine where this morphological
distribution was not seen. The present study was undertaken further to investigate
axoplasmicflowin toxic neuropathies.
AXOPLASMIC FLOW IN AXONAL NEUROPATHIES
237
RESULTS
Control Animals
The distribution of radioactivity in the L7 dorsal root and the sciatic nerve of
normal animals sacrificed at six hours and thirty days after dorsal root ganglion
injection is shown in fig. 1. Each point is the average of three animals. The lines
have been arbitrarily fitted to the points by eye, and are simply intended to aid in
the interpretation of the graphs. At six hours, the activity in the dorsal root ganglion
was much higher than at thirty days. At the earlier time in the sciatic nerve a peak
of radioactivity occurred about 80 mm from the ganglion and a front of radioactivity
about 120 mm from the ganglion. The peak may therefore be interpreted as being
due to material moving with a velocity of 320 mm/day, and the front with a speed
of about 480 mm/day. In other animals killed at shorter intervals than six hours,
a peak and front advancing at approximately these rates were seen, similar to those
described by Ochs and Ranish (1969). At thirty days a similar peak about 45 mm
from the ganglion was seen corresponding to material moving with a speed of about
1-5 mm/day. Similar curves were found by Lasek (1968). The sciatic nerve from
animals killed ten days after injection showed either no peak or only a very small
one. Lasek (1968) similarly found only a vague peak in animals sacrificed ten days
after dorsal root ganglion injection. The analysis in this paper is based upon the
movement of demonstrable waves of activity, and therefore the animals sacrificed
at ten days will not be discussed further.
Assessment of the individual speeds in each control animal and the height of the
peak are shown in Table I.
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was covered with sterile gelatine foam, the area sprayed with a polymyxin-bacitracin-neomycin
aerosol, and the skin closed with clips. The animals recovered quickly and on the day after
operation were able to walk normally. None developed neurological abnormalities as a result
of the operation. Animals were killed by an overdose of pentobarbitone six hours, ten days
and thirty days after dorsal root ranglion injection. Three animals at each time point from each of
the control, vincristine, acrylamide and TOCP groups were studied. In a standard manner the
seventh dorsal root was removed in continue with the injected L7 ganglion, the whole of the sciatic
nerve and its major branches to the level of the popliteal fossa. The nerve was laid on a metal
ruler marked in one mm lengths, frozen with solid carbon dioxide, and cut with a razor blade into
3 mm segments. Each was dissolved in low potassium scintillation vials, and the radioactivity
measured as previously described (Bradley, Murchison and Day, 1971). Statistical analysis of
groups was made using the Mann-Whitney U Test.
Material taken from the opposite hind-limb was used for histological study. The spinal cord,
dorsal and ventral roots, the sciatic nerve in the thigh, the posterior tibial nerve and digital nerves
in the foot were examined in paraffin sections stained with hasmatoxylin and eosin, solochrome
cyanin, and by the Weigert-Pal and Glees and Marsland methods. The hamstring, gastrocnemius,
and plantar muscles were studied in cryostat sections stained for myosin ATPase, NADH diaphorase,
and by the method of Namba and Grob; and in paraflBn sections stained with hffimatoxylin and
eosin, phosphotungstic acid-hasmatoxylin, and the Picro-Mallory method. The histological
severity of the neuropathy was graded into mild, moderate and severe. The clinical abnormalities
at the time of operation and of sacrifice were similarly graded.
238
W. G. BRADLEY AND M. H. WILLIAMS
5.10"
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distance along sciatic nerve - mm.
FIG. 1.—Distribution of radioactivity within 3 mm segments of L7 dorsal root ganglia and
sciatic nerve of control cats sacrificed six hours and thirty days after injection with 3H-leucine.
Each point is the average of values derived from three animals.
TABLE I.—DATA ON AXOPLASMIC FLOW AT SIX HOURS A N D THIRTY DAYS AFTER L7 DORSAL ROOT
GANGLION INJECTION IN CONTROL ANIMALS A N D THOSE WITH TOXIC NEUROPATHIES
Time
6 hour
Croup
Control
Viocristino
Acrylamide
TOCP
30 day
Control
Vincristinc
Acrylamide
TOCP
Crest
dpm
1203
2655
1250
736
936
2078
1350
3800
3050
1600
1675
325
569
1925
2693
165
3910
1246
4622
2750
1800
2500
2000
2200
Ganglion
dpm
444,800
418,700
240,000
491,600
559,100
397,500
500,000
500,000
250,000
320,000
290,000
214,000
6,700
12,100
20,000
9,000
10,300
9,900
23,200
18,000
16,000
13.000
11,200
30,000
Crest/
ganglion
per cent
0-271
0-63 Y
O-52J
om
0-17 Y
0-52 J
0-27 "1
O76t
1-22J
0-50T
0-58 i1-52J
8-5 "
8-5-)
15-9
5-9 {•
3-5 J
37-9
12-6
16-41
15-3 111-3J
19-3")
9-3")
17-9
7-9 J-
I7-3J
7-3J
A verage
crest]
ganglion
per cent
0-47
0-28
0-75
0-87
12-6
17-5
14-3
14 8
Crest rate
mrnjday
364 "I
432^
360 J
2531
288 J288 J
265")
324^
324 J
3281
3 0 0 )•
312J
Average
crest rate
mmlday
383
304
313
1-4
1-4-1
1-2 [
1-2J
0-3 "1
0-9 V
0-8 J
Average
front rate
mm/day
459
3041
276
1-7J
0-8J
Front rate
mm/day
4281
456 Y
492 J
1-2
1-3
0-9
3 2 4 )•
436J
3241
504 Y
420 J
4441
372 V
420 J
355
416
412
Downloaded from http://brain.oxfordjournals.org/ by John Lyftogt on September 11, 2013
E
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239
AXOPLASMIC FLOW IN AXONAL NEUROPATHIES
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distance along sciatic nerve • mm
FIG. 2.—As fig. 1 for animals with vincristine neuropathy.
Downloaded from http://brain.oxfordjournals.org/ by John Lyftogt on September 11, 2013
Animals with Toxic Neuropathies
The distribution of radioactivity in the sciatic nerves of these animals are shown
in figs. 2, 3 and 4. Again each point corresponds to the average of 3 animals. In
acrylamide neuropathy at six hours and in vincristine neuropathy at thirty days,
there was a considerable scatter of points which may correspond to a number of
minor peaks of axoplasmic flow, or may be due to experimental error or biological
variation. Nevertheless, at the two time points in each group, a wave of activity
was clearly discernible, indicating that axoplasmic flow occurred in all three groups of
animals with toxic neuropathies. The speeds and the crest heights recorded in each
individual animal are shown in Table I.
Crest heights.—There is wide variation between animals in all four groups. Thus
at six hours in the normal animals, the crest height expressed as a percentage of the
dorsal root ganglion activity ranged from 0-27-0-52 per cent, while in animals with
vincristine neuropathy studied at this time, one value was comparable to normal,
and two values were below the normal levels; similarly at thirty days, the crest height
in normal animals varied from 8-5-15-9 per cent of the dorsal root ganglion level,
and in the vincristine animals there was one very low value of 1-8 per cent and one
very high value of 37-9 per cent. There is clearly no significant difference between the
mean crest heights either of the "slow" or "fast" waves in these four groups of
animals, though the average height of the "fast" wave crest is somewhat lower in
animals treated with vincristine than in the controls.
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10=
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60
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distance along sciatic nerve mm.
60
100
140
distance along sciatic nerve • mm.
FIG. 3.—As fig. 1 for animals with acrylamide neuropathy.
j.10°
Acrylamide 6hours
10*
TOCP 30days
£ 10;
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distance along sciatic nerve • mm.
FIG. 4.—As fig. 1 for animals with TOCP neuropathy.
Downloaded from http://brain.oxfordjournals.org/ by John Lyftogt on September 11, 2013
co
AXOPLASMIC FLOW IN AXONAL NEUROPATHIES
241
DISCUSSION
The most important conclusion to be drawn from this study is that axoplasmic
flow may be shown to occur in a retrograde direction down the sensory nerve fibres of
cats with vincristine, acrylamide or TOCP neuropathy. Peaks of radioactivity were
seen in the sciatic nerves both at six hours and thirty days in similar positions to those
found in normal animals. Even though only three time points were studied, the
fact that the peak at six hours after injection of 3H-leucine into the dorsal root
ganglion was not seen at ten days, and that a new peak had appeared at thirty days
may be taken as clear evidence of the presence of axoplasmic flow (Lasek, 1968;
Ochs and Ranish, 1969).
We have therefore been unable to substantiate the rinding of Pleasure et al.
(1969) of no axoplasmic flow in feline sensory roots in acrylamide neuropathy, but
to confirm that flow may occur in TOCP neuropathy; Pleasure et al. (1969) studied
the dorsal root, that is, axoplasmic flow in the orthodromic direction. The findings
described in this report relate to the flow along the part of the nerve usually showing
the "dying back" phenomenon, that is, the peripheral branches of the sensory fibres.
In normal animals, like Lasek (1968), we did not find evidence of moving peaks of
radioactivity in the dorsal root. Though Pleasure et al. (1969) used autoradiography,
they measured the total endoneurial radioactivity, and so like us were unable to define
just how much extra-axonal radioactivity was being measured. One refinement of the
method would have been to measure autoradiographically only axoplasmic
radioactivity as in the study of Bradley and Jaros (1973), but as moving peaks of
radioactivity were seen both in the present study and in that of Pleasure et al. (1969)
it seems likely that the majority of the activity being studied was in fact intra-axonal.
The question which remains therefore is whether there is any quantitative difference
between the flow of material in cats with neuropathies and normal cats. Within
each toxic group there was marked variation in the height of the waves of axoplasmic
flow, and no significant difference from normal was demonstrated between the
heights of both the "fast" and "slow" waves. The results therefore give no support
Downloaded from http://brain.oxfordjournals.org/ by John Lyftogt on September 11, 2013
Crest and front velocities.—In this analysis there was less variation between
animals of a group. The crest velocity in all the cats with toxic neuropathies sacrificed
six hours after 3H-leucine injection, and in all the TOCP-treated cats sacrificed thirty
days after injection was significantly below that in the normal animals (P=0-05).
The velocity of the crest of slow axoplasmic flow in vincristine- and acrylamidetreated animals was not different from that in control animals. The velocity of the
front of both "fast" and "slow" moving radioactivity did not differ significantly
from normal in any group other than in the TOCP-treated animals sacrificed at
thirty days after injection of the ganglion (P=005). The reduction of mean velocity
for the "fast" wave in each group ranged from 19 per cent in TOCP-treated animals
to 28 per cent in vincristine neuropathy. The "slow" wave in the cats with TOCP
neuropathy was slowed by 36 per cent compared with normal.
242
W. G. BRADLEY AND M. H. WILLIAMS
Downloaded from http://brain.oxfordjournals.org/ by John Lyftogt on September 11, 2013
to the hypothesis that a decrease in the amount of the axoplasmic flow of protein is
the cause of axonal degeneration in toxic neuropathies.
On the other hand, significant differences from normal in the velocities of the
crests, but not of the fronts, of these waves, were found. The crests of the "fast"
wave in all the neuropathic animals, and of the "slow" wave in the TOCP-treated
animals moved more slowly than normal.
The decrease in the rate of delivery of protein at fast rates of flow in vincristine
neuropathy is interesting. Vincristine and vinblastine, like colchicine, bind to
neurotubular protein and produce neurofibrillary accumulation in the nerve cell
(Schochet, Lampert and Earle, 1968; Wisniewski, Terry and Hirano, 1970; Seil
and Lampert, 1968; Journey, Burdman and Whaley, 1968; Krishan and Hsu, 1969;
Olmsted and Rosenbaum, 1969; Marantz and Shelanski, 1970). Both colchicine
and vinblastine impair fast and slow axoplasmic flow (Dahlstrom, 1968; Fernandez,
Huneeus and Davison, 1970; Fernandez, Burton and Samson, 1971) though
colchicine affects the fast more than the slow rates of flow (Karlsson and
Sjostrand, 1969; Sjostrand, Frizell and Hasselgren, 1970). The intraneural injection
of colchicine (Angevine, 1957), and the systemic administration of vincristine
(Bradley et ah, 1970; Bradley, 1970) produce axonal degeneration. It was for these
reasons that Bradley et ah (1970) suggested that vincristine, which does not produce
a "dying back" picture, might cause a decrease in the fast rates of axoplasmic flow,
and the "dying back" neuropathies a decrease in the slower rates of flow. The present
study supports this suggestion with regard to vincristine, though the situation in the
"dying back" neuropathies is more complex. In TOCP neuropathy both "fast"
and "slow" crest velocities were reduced, while in acrylamide neuropathy the "fast"
but not the "slow" crest was retarded.
There was no correlation between the rates and amounts of axoplasmic flow, and
the clinical or histological severity of the neuropathy. It is possible that the clinical
signs may in part have been complicated by central nervous degeneration which
occurs to some extent in acrylamide and TOCP intoxication (Fenton, 1955; Prineas,
1969a and b). If, however, the hypothesis that axonal degeneration is due to
impairment of axoplasmic flow of protein were correct, the more severely affected
animals would have been expected to have the greater impairment of flow, and this
was not so.
In this study no attempt was made to measure the specific radioactivity of purified
protein from each nerve segment. However as argued elsewhere (Bradley et al.,
1971), within three hours of injection the major part of 3H-leucine will have been
synthesized into protein. Only a minor part is incorporated into other cell components
including lipid. Since very few lipid droplets are to be found within the axoplasm
on electronmicroscopy, the major part of axonal lipid is presumably phospholipid.
The axonal flow of phospholipid is small in amount, and occurs without evidence
of moving peaks of radioactivity (Miani, 1964,1967). It is therefore unlikely that the
presence of components other than protein will have interfered with the analysis
made here.
AXOPLASMIC FLOW IN AXONAL NEUROPATHIES
243
SUMMARY
A study of waves of "fast" and "slow" axoplasmic flow of radioactivity following
the injection with 3H-leucine of the L7 dorsal root ganglion of cats suffering from
toxic neuropathies is presented. Comparison was made between normal animals,
and those intoxicated with vincristine, acrylamide and triorthocresyl phosphate.
The heights of these waves in the intoxicated animals were normal. The rate of
movement of the crests of the "fast" waves, but not of the fronts, was reduced in all
experimental groups. The velocity of the "slow" wave was reduced only in TOCP
neuropathy.
It is argued that this reduction of velocity is unlikely to be the cause of the axonal
degeneration. The complexity of investigations of axoplasmic flow in toxic
neuropathies is highlighted.
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We must now discuss the significance of the demonstrated decrease in velocity of
normal amounts of material with regard to axonal degeneration in toxic neuropathies.
If the hypothesis advanced in the introduction were correct, the amount of protein
delivered to the distal parts of the nerve might be the significant factor in the
maintenance of axonal integrity. It therefore becomes a four-dimensional problem
involving both the rate of flow and the amount of material transported. There are
insufficient data available from the present study to build up a clear picture of the
arrival of material in the nerve terminals in these animals. Nevertheless, at the most
the delivery of material was reduced by 36 per cent, and was probably very much
less affected than this. It seems unlikely that a biological process would have such a
small safety factor that perhaps a 10 per cent reduction would cause structural
breakdown. We therefore do not believe that the changes demonstrated here could
cause the axonal degeneration demonstrated.
One possibility, which cannot however be excluded, is that axonal integrity depends
upon a protein constituting only a minor fraction of axoplasmic flow. Quantitative
fractionation of these proteins in disease states would be of value.
The study of axoplasmic flow in toxic neuropathies is by no means as simple as
might at first appear. In most neuropathies degeneration and to a greater or lesser
extent regeneration occur side by side. A totally degenerated axon will not transport
protein. As discussed in the following paper (Bradley and Jaros, 1972), axoplasmic
flow may be increased in regenerating fibres. All studies must face this problem,
particularly those where a single application of a toxin is used.
Another complexity is that the moving waves used in this study constitute only a
minor part of the total axoplasmic flow. As Bradley et al. (1971) showed, most of
the material moves in a graded manner, the amount of the fast flowing material
being much less than that of the slow. A full analysis of axoplasmic flow requires
the quantification of the amount of material moving at all velocities, by a method such
as that of velocity spectrum analysis described by Bradley et al. (1971). A large
number of animals are required to allow such an analysis, and to date no such
analysis has been attempted.
244
W. G. BRADLEY AND M. H. WILLIAMS
ACKNOWLEDGMENTS
This study was supported by grants from the Muscular Dystrophy Group of Great Britain, the
Medical Research Council, the Muscular Dystrophy Associations of America, and the National
Fund for Research into Crippling Diseases. We are grateful to Messrs. Coalite and Chemical
Products Ltd. for supplying the triorthocresyl phosphate, and to Mr. Paul Clarke of the Statistical
Section of the Newcastle Regional Hospital Board for the statistical analysis.
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